Lysosomal Cholesterol Sets the Autophagy Triage Threshold by Tuning mTORC1 Reactivation

Hypothesis

The selectivity of induced autophagy is not solely governed by nutrient‑sensing kinases; it is additionally calibrated by lysosomal membrane cholesterol, which modulates the rate of mTORC1 reactivation on the lysosomal surface. When lysosomal cholesterol is elevated, mTORC1 reassociates more rapidly with Rag GTPases, shortening the window of AMPK‑driven ULK1 activation and biasing the cell toward bulk, less selective macroautophagy. Conversely, reduced lysosomal cholesterol prolongs the AMPK‑ULK1/TFEB signaling window, enhancing selective cargo recognition via receptors such as p62 and NBR1 and favoring chaperone‑mediated autophagy (CMA) for KFERQ‑motif proteins. This predicts that manipulating lysosomal cholesterol will shift the autophagy triage point, altering which substrates are spared during famine.

Mechanistic Basis

Lysosomal cholesterol influences the physicochemical properties of the lysosomal membrane, affecting V‑ATPase activity and the luminal pH that regulates Ragulator‑Rag signaling. High cholesterol stabilizes a more ordered lipid environment, facilitating Rag GTPase loading and thus faster mTORC1 docking after ATP depletion. Low cholesterol increases membrane fluidity, delaying Rag‑mediated mTORC1 recruitment and sustaining AMPK activity. This creates a feedback loop where the lipid state of the lysosome sets the duration of the autophagy‑inducing signal, effectively acting as a rheostat for the triage decision between selective (CMA, mitophagy) and non‑selective bulk degradation.

Predictions

1. In neurons subjected to 12‑hour fasting, pharmacological depletion of lysosomal cholesterol (using methyl‑β‑cyclodextrin) will increase the proportion of KFERQ‑containing proteins degraded via CMA, measured by immunoblot for GAPDH and LC3‑II colocalization with LAMP2A.
2. The same cholesterol depletion will reduce nonspecific degradation of cytosolic markers (e.g., long‑lived proteins labeled with puromycin‑associated nascent chain proteomics) without altering total autophagic flux (LC3‑II turnover).
3. Conversely, lysosomal cholesterol enrichment (via U18666A treatment) will accelerate mTORC1 reactivation (phospho‑S6K rebound) after refeeding, leading to earlier cessation of ULK1 phosphorylation and a shift toward bulk autophagy, detectable by increased non‑selective proteasome‑independent degradation of mitochondrial matrix proteins.
4. Genetic knockdown of NPC1, which causes lysosomal cholesterol accumulation, will blunt the fasting‑induced increase in selective autophagy receptors and exacerbate synaptic loss in hippocampal slices, linking the mechanism to maladaptive autophagy.

Experimental Approach

• Culture primary hippocampal neurons; treat with MβCD (cholesterol depletion) or U18666A (cholesterol loading) for 2 h before initiating HBSS‑based fasting.
• Monitor autophagic flux with bafilomycin A1 chase and LC3‑II/Western blot.
• Quantify CMA activity using a KFERQ‑GFP reporter and assess selective mitophagy via mt‑Keima.
• Measure mTORC1 reactivation timing by phospho‑S6K immunoblotting at 0, 30, 60 min post‑refeeding.
• Perform proteomic SILAC labeling to distinguish selective versus bulk protein degradation.
• Validate findings in NPC1‑knockdown neurons and assess synaptic marker density (PSD‑95, synaptophysin) after prolonged fasting.

If cholesterol modulation reliably shifts the autophagy selectivity window as predicted, the hypothesis will be supported; lack of measurable changes in cargo preference despite altered lysosomal cholesterol would falsify the core claim that lysosomal lipid composition sets the triage threshold.